Photo-labile compounds, their synthesis and use as fluorophores

ABSTRACT

##STR1## Compounds of formulae (I), (II) wherein X is an optionally substituted benzyl group which carries an NO 2  group in the ortho-position, Z is a group of formula α or β, R, R&#39; and Y are each optionally substituted groups, R&#34; is hydrogen or alkyl, m and n are each integers of from 1 to 6, and q is an integer of from 1 to 20, and salts thereof. These protected or caged organic compounds, based on the dyes fluorescein (I) and rhodamine (II), can be introduced into biological systems and there released by means of light radiation. They are suitable, for example, for use in the labelling of proteins or lipophilic structures. Processes for the preparation of the compounds are also described.

This application is a 371 of PCT/GB91/01941 filed Nov. 6, 1991.

This invention relates to novel protected or "caged" organic compounds,based on the dyes fluorescein and rhodamine, which can be introducedinto biological systems and there released by means of light radiation.These photo-labile compounds, or photoactivatable fluorophores, aresuitable for use in labelling proteins. Processes for the preparation ofthe compounds are also described.

The study of the structure and dynamics of cellular and other biologicalsystems by the attachment of fluorescent labels to molecular species isa well known and widely used approach. A technique which has beenreported in recent years is fluorescence photoactivation and dissipation(FPD). This technique was first described by Ware et al. in Applicationsof Fluorescence in the Biomedical Sciences, 141-157, 1986, Edited byTaylor et al., Liss, New York. The basic feature of an FPD measurementis the attachment of a molecular label that is initially non-fluorescentbut which will become fluorescent when exposed to a short pulse of lightof the appropriate wavelength. Such molecules are calledphotoactivatable fluorophores (PAF).

Krafft et al., J. Am. Chem. Soc., 1988, 110, 301-303, describephotoactivatable fluorophore compounds based on difunctionalisedfluoresceins. The development of such compounds was prompted by the factthat fluorescein can exist as either of two tautomeric forms: afluorescent, xanthen-3-one form or a non-fluorescent, lactone form. Thecompounds are held in the non-fluorescent lactone tautomer throughdialkylation of the two phenolic oxygens, with one of the ether groupsbeing susceptible to a photo-cleavage reaction that triggers the openingof the lactone structure to produce the fluorescent xanthen-3-onetautomer.

The invention shows that the fluorescence of rhodamine, on the otherhand, is quenched by introducing electron-withdrawing groups onto theamine groups, thereby reducing electron delocalisation throughout thearomatic system.

Mitchison, The Journal of Cell Biology, 109, 1989, 637-652, describesthe use of a derivative of carboxyfluorescein, the synthesis of which isbased on the work of Krafft et al. (op. cit.). The compound isnon-fluorescent, but can be converted to a fluorescent form by exposureto light of 365 nm wavelength. This photoactivatable fluorescent probewas covalently attached to a globular protein, tubulin, andmicro-injected into mitotic cells in tissue culture (where itincorporated into functional spindles).

Notwithstanding the above, the compounds described by Krafft et al. andby Mitchison suffer from certain disadvantages which includedifficulties in synthesis. For example, the methods for theirpreparation do not permit clean substitution of single photo-sensitivegroups on one of the two phenolic oxygens and this results in the needfor extensive chromatographic purification. This is an important pointbecause the presence of as little as 1% of free fluorophore in thephotoactivatable compound is sufficient to seriously restrict itseffective use in biological research. The compounds described by Krafftet al. and by Mitchison also suffer from a relative lack of watersolubility, which is an important property when considering thelabelling of biological molecules. Furthermore, the compounds of Krafftet al. and of Mitchison are all based on fluorescein. It will beappreciated that a range of probes with differing optical propertieswould be desirable.

There is therefore a need for further compounds suitable for use asphotoactivatable fluorophores and which have more controlled synthesesincorporating crystalline intermediates and also having improvedproperties, such as water solubility and the ability to bind at specificsites on proteins, or a lipophilic nature and the ability to bind tolipid structures.

The present invention seeks to provide such compounds and processes fortheir preparation.

According to the present invention there are provided compounds of thefollowing formulae (I) and (II): ##STR2## wherein X is an optionallysubstituted benzyl group which carries a NO₂ group in theortho-position,

Z is a group of the formula: ##STR3##

R, R' and Y are each optionally substituted groups,

R" is hydrogen or alkyl, and

m and n are each integers of from 1 to 6,

q is an integer of from 1 to 20, and salts thereof.

Groups R, R' and Y are each optionally substituted and this permits theintroduction of water-solubilising and protein-reactive functions intothe compounds. The person skilled in the art will readily appreciate thevariety of substituent groups available and the criteria for selectingthose which will impart the desired properties or characteristics to thefinal compound. For example, groups R, R' and Y may be lower (C₁ -C₆)alkyl chains substituted individually or severally with ionisablefunctions such as carboxylate or sulphonate, and/or with functionsspecific for protein labelling such as maleimides, haloacetates orreactive esters. The multifunctional side chain shown in structure (I)represents one such example. R" is hydrogen or alkyl, most typicallylower (C₁ -C₆) alkyl.

The compounds (I) and (II) are photocleavable and are useful asphotoactivatable fluorophores. It will be appreciated that thefluorophore comprises fluorescein in compound (I) and rhodamine incompound (II). It will be further appreciated that, depending upon theidentity of the sidechain of substituent Z, the compounds (I) willexhibit either good water solubility or strong lipophilic properties,and this will obviously influence their areas of use.

A particularly preferred photoactivatable compound of this invention hasthe following formula: ##STR4##

According to the present invention there is further provided a methodfor preparing the compounds (I) and which comprises the followingsequence of reactions: ##STR5##

In an alternative approach, suitable where the fluorophore constituentis rhodamine, the invention provides a method for preparing thecompounds (III) and which comprises the following sequence of reactions:##STR6##

The intermediate compound of the following formula, referred to in theabove reaction sequence, is a novel compound and is a further aspect ofthis invention: ##STR7##

A still further aspect of the invention is a method for preparing thecompound (7) and which comprises the steps shown in the followingReaction Sequence 1. ##STR8##

The compounds (I) and (II) of this invention are photocleavable and finduse as photoactivatable fluorophores for the labelling of, for instance,proteins and lipid structures. For example, they can be used to labelproteins at the site of a cysteine side chain. Such a labelled proteinis introduced into a biological system, and is there photoactivated soas to form the fluorescent compound in situ in the biological system.The fate of the fluorescent signal is then followed. Procedures forachieving the introduction, such as micro-injection techniques, are wellknown and need not be described here in detail.

The conditions required for photolysis of the compounds are the same aswith conventional photocleavable ortho-nitrobenzyl compounds. Lighthaving a wavelength of from 300 to 350 nm is suitable for this purposesuch as may be generated by a xenonarc flash lamp or a 347 nm frequencydoubled ruby laser. Longer exposure from a filtered (300 to 350 nm)mercury or xenon-arc lamp source is perfectly adequate when high timeresolution is not required. The photoactivatable fluorophore may beilluminated through a microscope in order to achieve appropriate spatialresolution within the biological system.

The invention thus provides a range of new compounds suitable for use asphotoactivatable fluorophores and which are synthesised through the useof a novel intermediate compound (7). The compounds exhibit good watersolubility, typically have a simple and specific reaction with the thiolgroup of the cysteine side chain in protein molecules and possessspectroscopic properties desirable for their intended use. Certain ofthe compounds of formula (I) exhibit highly lipophilic properties andare thus suited for use in the labelling of lipid structures. The methodby which they are formed is relatively clean, efficient and producesgood yields.

The compounds (I) and (II) of the invention are designed for use inbiological studies and are, for example, ideally suited for use in theaforementioned fluorescence photoactivation and dissipation (FPD)technique. As will be appreciated, once a label has been introduced intoa biological system, it is possible to follow the movement and functionof the labelled molecules, typically proteins, and their interactionwith other components of the cell system. It has previously been shownthat proteins labelled with fluorophores will be incorporated into thefilamentous proteins characteristic of the internal cell structure andthis can provide useful information about cell structure and the cellcycle (e.g. mitosis). With the compounds (I) and (II) of the invention,there is the additional advantage that the fluorescent label can bereleased at a localised point in the cell and the fate of the labelledprotein can then be followed as a function of time. This allows theinvestigation of spatial movement and diffusion rates in a cellularsystem.

The compounds and method of this invention will now be furtherillustrated by the following Examples. Examples 1 and 3 relate to thepreparation of a caged fluorescein compound of the invention; Example 2refers to the synthesis of the novel intermediate compound used in thispreparation. Example 4 demonstrates the labelling of a protein thiolwith the caged fluorescein compound. Example 5, which is included toillustrate the preparation of compounds based on rhodamine, refers to amodel of the chemistry in which the acyl group on one of the amines isacetyl (i.e. it blocks the fluorescence by electron withdrawal, but isnot photocleavable). Example 6 relates to the synthesis of a cagedfluorescein compound of the invention and which has a highly lipophilicnature.

EXAMPLE 1

Fluiorescein monoallyl ether (2) was prepared by hydrolysis offluorescein allyl ether ester (1) using a modification of the method ofHurd & Schmerling¹. Thus the ether ester (1; 2 g) dissolved in hotacetone (100 ml) was treated with 1.25M NaOH (60 ml) and the solutionwas heated under reflux for 12 minutes, then poured into water (200 ml).The solution was acidified with concentrated HCl and the precipitatedsolid was filtered, washed with water and recrystallised from ethanol toafford the monoether (2) as yellow crystals (0.83 g, 46%), m.p. 206°-7°C. (lit.¹ 205° C.).

3'-O-(2-Nitrobenzyl) -6'-O-allylfluorescein [(3) where X=2-nitrobenzyl].The preparation of this compound was based on the method of Krafft,Sutton & Cummings.² Thus a suspension of the allyl ether (2; 0.80 g) indry benzene (14.5 ml) and dry tetrahydrofuran (4.5 ml) was treated with2-nitrobenzyl bromide (0.746 g) and silver (I) oxide (1.02 g) andstirred under reflux in the dark for 16 hours. The mixture was cooled,filtered and the precipitate washed with ethyl acetate. The combinedfiltrates were evaporated and the residue purified by flashchromatography (Merck*9385 silica gel) using CH₂ Cl₂ -petroleum ether(75:25) as the eluting solvent. Fractions containing the product werecombined, the solvent was evaporated and the residue was triturated withether to afford the product (3) as a colourless solid (0.68 g; 62%),which crystallised from ethyl acetate-petroleum ether as plates, m.p.171°-2° C. (Found: C, 71.1; H, 4.2; N, 2.7. C₃₀ H₂₁ NO₇ requires C,71.0; H, 4.2; N, 2.8%).

EXAMPLE 2

t-Butyl N'-(t-Butoxycarbonylmethyl)hydrazinecarboxylate [(4) wherem,n=1]. The compound was prepared by modification of the method ofStreicher and Reinshagen³ for the corresponding ethyl ester. Thus asolution of t-butyl carbazate (5.28 g), t-butyl bromoacetate (7.80 g)and triethylamine (4.06 g) in dry benzene (40 ml) was heated underreflux for 16 hours, cooled and filtered. The filtrate was washed withsaturated NaHCO₃ and brine, dried and evaporated. The residue wasdissolved in ethanol (85 ml) which contained pyruvic acid (4.4 g) and 4MNaOAc (11.5 ml) was added. After standing for 1 hour at room temperaturethe mixture was diluted with ether, washed with saturated NaHCO₃ andbrine, dried and evaporated. The residue was distilled to give theproduct (4) as a colourless viscous liquid (2.8 g; 28%) whichcrystallised on standing, m.p. 48°-9° C. (Found: C, 54.0; H, 9.3; N,10.95. C₁₁ H₂₂ N₂ O.sub. 4 requires C, 53.6; H, 9.0; N, 11.4%).

t-ButylN'-(t-Butoxycarbonylmethyl)-N'-(2-(2,5-dihydro-2,5-dioxopyrrol-1-yl)acetyl)hydrazinecarboxylate[(6) where m,n=1]. A suspension of2,5-dihydro-2,5-dioxopyrrol-1-ylacetic acid (821 mg) in thionyl chloride(15.9 ml) was heated under reflux for 0.5 hours and the excess thionylchloride was removed under vacuum. The residue was evaporated twice withdry toluene to afford 2,5-dihydro-2,5-dioxopyrrol-1-ylacetyl chloride[(5) where m=1] as a colourless, readily hydrolysed liquid. (Thecompound has been prepared previously by a less convenient route⁵). Thecrude acid chloride was dissolved in dry ether (25 ml) and addeddropwise to an ice-cold solution of the hydrazinecarboxylate [(4) wheren=1, 1.30 g] and triethylamine (588 mg) in dry ether (25 ml). Themixture was stirred in an ice bath for 1 hour then diluted with ethylacetate and washed with water, dilute HCl, saturated NaHCO₃ and brine,dried and evaporated. The residue was crystallised from ethylacetate--petroleum ether to give the compound (6) as colourless plates(1.67 g; 82%) m.p. 157°-9° C. (Found: C, 53.1; H, 6.7; N, 10.8. C₁₇ H₂₅N₃ O₇ requires C, 53.25; H, 6.6; N, 11.0%).

N-Carboxymethyl-2(2,5-dihydro-2,5-dioxopyrrol-1-yl)acetyl hydrazidehydrochloride [(7) where m,n=1].

The compound (6; 400 mg) was dissolved in trifluoroacetic acid (1.5 ml)and kept at room temperature for 1 hour. The trifluoroacetic acid wasevaporated under reduced pressure and the residue was redissolved in a2M solution of anhydrous HCl in dioxan (4 ml). Colourless crystals beganto separate after a few minutes and the solution was diluted with dryisopropyl ether (10 ml), filtered and the precipitate washed withisopropyl ether and dried to give the hydrochloride (7) which contained0.5 equivalent dioxan of solvation (256 mg, 79%).

EXAMPLE 3

Caged fluorescein-maleimide [(9) where m,n=1, X=2-nitrobenzyl]. Asolution of the 2-nitrobenzyl allyl fluorescein (3; 51 mg) in CH₂ Cl₂(10 ml) and MeOH (0.2 ml) was cooled to -50° C. and treated with astream of ozonised oxygen until t.l.c. analysis (silica gel; ethylacetate--petroleum ether 1:1) showed complete consumption of thestarting material (ca. 30 minutes). The solution was purged withnitrogen, treated with dimethyl sulphide (0.10 ml) and allowed to warmto room temperature over 1 hour, then stirred for a further 1 hour,diluted with CH₂ Cl₂, washed with water and brine, dried and evaporated.The residue was purified by flash chromatography (ethyl acetate--lightpetroleum 1:1) to afford the aldehyde [(8) where X=2-nitrobenzyl] as apale gum (36 mg) which was characterised by ¹ H-NMR spectroscopy. Thismaterial was dissolved in dimethyl formamide (0.2 ml) and treated with asolution of the hydrazide hydrochloride [(7) where m,n=1; 26 mg] in EtOH(0.3 ml) and 2M NaOAc (0.06 ml). The solution was kept in the dark for 2hours at room temperature then diluted with ethyl acetate and washedwith 1M citric acid and brine, dried and evaporated. The residue waspurified by flash chromatography (ethyl acetate-methanol-acetic acid95:5:0.5) to give the product (9) as a pale gum (24 mg), which wascharacterised by ¹ H NMR. The material was stored in the dark at 4° C.as a dilute solution in EtOAc.

After addition of 2-mercapto ethylsulphonate to the maleimide group, thequantum yield for photolysis was 0.65 (irradiation 300-350 nm) and therate of release of the fluorophore was 7s⁻¹, both measurements being atpH 7.0 and 22° C.

EXAMPLE 4

Labelling a protein thiol with caged fluorescein-maleimide (9). Myosinlight chain-1 (LC-1) was dissolved at 1 mg/ml in a solution of 50 mMTris-HCl/2 mM EDTA/2 mM dithiothreitol at pH 7.5 and kept for 15 minutesthen dialysed for 2 hours against 10 mM PIPES/2 mM EDTA/2 mM ascorbate,pH 6.5. One aliquot, A, (1 ml) was treated with an aliquot (0.10 ml) ofa solution containing 400 mM sodium phosphate/2 mM ascorbate/8 mMN-ethylmaleimide, pH 7.5 and incubated for 20 minutes at roomtemperature. A further aliquot, B, (1 ml) was treated with aliquot (0.10ml) of a solution containing 400 mM sodium phosphate/2 mM ascorbate, pH7.5 and incubates A and B were then immediately treated with aliquots(0.10 ml) of a solution of 8 mM caged fluorescein-maleimide [(9) whereX=2-nitrobenzyl, m,n=1] in dimethyl formamide. Each solution was keptfor 30 minutes in the dark at room temperature, then excess reagent wasquenched by addition of aliquots (50 μl) of 40 mM mercaptoethanol in 10mM Tris-HCl, pH 7.5. Each incubate was dialysed for 2 hours in the darkagainst 10 mM Tris-HCl, pH 7.5 and subjected to gel filtration onSephadex* G50 (1 g dry weight) in the same buffer. Fractions containingthe protein were pooled and aliquots were irradiated (300-350 nmwaveband) until maximum release of fluorescence was observed (ca. 30seconds with a 100 W lamp). The irradiated solutions were analysed bySDS-polyacrylamide gel electrophoresis (approx. 2 μg labelled proteinper track) and the gel was photographed under ultraviolet illuminationto detect fluorescent bands. Subsequent staining with Coomassie Blueidentified the position of the myosin light chain-1. FIG. 1 shows theexperimental result and reveals the presence of significant quantitiesof free dye, i.e. not covalently bound to the protein and dissociatingduring the electrophoresis run. Further processing of the cagedfluorescein-labelled protein by f.p.l.c. on a Mono Q column (Pharmacia)gave fractions which when analysed as above were entirely free ofnon-covalently bound dye.

EXAMPLE 5 (see Reaction Sequence 2)

3-Amino-6-diethylamino-9-(2-methoxycarbonylphenyl) xanthylium chloride(11). The synthesis of unsymmetrical rhodamines followed longestablished methods, e.g. Ref. 6 and references therein. A mixture of3-aminophenol (0.57 g), 2-(2-hydroxy-4-diethylaminobenzoyl)benzoic acid(1.5 g) and concentrated sulphuric acid (9 ml) was heated at 105° C. for4 h, cooled and poured slowly into an ice cold solution of KOH (18.9 g)in water (50 ml). The solution was adjusted to pH 2-3 with dilute KOHand the precipitate was filtered and dried in vacuo. The dried solid wasdissolved in MeOH, filtered from inorganic salts and evaporated to leavethe crude rhodamine (10) as a dark solid (1.92 g; 107%). This materialwas dissolved in MeOH (20 ml) and hydrogen chloride was bubbled into thesolution for 20 min, then for a further 3 h while the solution washeated at 60° C. The solution was concentrated to approx. 7 ml, dilutedwith water and extracted with CHCl₃. The organic extract was dried andevaporated to give the crude ester (11) as a dark solid (2.2 g; 105%),λ_(max) 531.5 nm (ε66,300).

3-Amino-6-diethylamino-9-(2-methoxycarbonylphenyl)xanthene (15).

Preparation of this compound was based on the method of Sensui, Gondaand Obara⁷. Thus a portion (0.50 g) of the crude ester (11) wasdissolved in MeOH (25 ml) and sodium borohydride was added in portionsover 2 h, until the initial dark red colour was discharged (total NaBH₄approx. 0.2 g). The solution was stirred for a further 1 h at roomtemperature, concentrated under reduced pressure to a small volume,diluted with water and extracted with chloroform. The extract was driedand evaporated to leave a pale gum (355 mg) which showed the presence ofthe xanthene (15) on t.l.c. (silica gel; benzene) as a colourless spot,R_(f) approx. 0.5, which rapidly turned red on exposure to light andair.

3-Acetamino-6-diethylamino-9-(2-methoxycarbonylphenyl)xanthene (16). Theaminoxanthene (15; 175 mg) was dissolved in CHCl₃ and treated withtriethylamine (450 mg) and acetyl chloride (175 mg). The solution wasstirred at room temperature for 1 h, evaporated to dryness and the majorcomponent isolated by flash chromatography, initially with ethylacetate--light petroleum (3:5) which eluted non-polar impurities,followed by chloroform which gave the acetylated product (16) as a palepink foam (102 mg; 52%) after removal of the solvent. Particularfeatures of the ¹ H NMR spectrum which confirmed the structure of theproduct were signals at δ 6.11 (s, 1H, H-9) and 2.09 (s, 3H, acetyl).

Analogous compounds were obtained when 2-nitrobenzyl chloroformate or1-(2-nitrophenyl)ethyl chloroformate were used in place of acetylchloride.

3-Acetimino-6-diethylamino-9-dehydro-9-(2-methoxycarbonylphenyl)xanthene(17). The preparation of this compound by tetrachlorobenzoquinoneoxidation was based on the method of Sensui, Gonda and Obara.⁷ Theacetamino compound (16; 100 mg) was dissolved in glacial acetic acid(0.5 ml) and tetrachlorobenzoquinone (100 mg; 1.8 equivalents) wasadded. The colour of the solution immediately turned to deep red.Chloroform (3 ml) was added and the solution was stirred for 1 h at roomtemperature, then further diluted with chloroform and washed withsaturated NaHCO₃, dried and evaporated. The residue was purified byflash chromatography in CHC₃ -EtOH 20:1, which eluted non-polarcomponents, followed by CHCl₃ -EtOH 10:1 and 4:1 to elute the product(17) which was obtained as a deep red solid (78 mg), λ_(max) 499,533 nm(ε 35,200 and 38,300). In the ¹ H NMR spectrum, the downfield shift ofthe acetyl signal (δ 2.40) as compared to its position in the reducedprecursor (16) was consistent with the proposed structure. ##STR9##

The relative fluorescence intensities of the compounds (11),[R,R',R"=Et, Y=H] and (17) were approximately 57:1.

EXAMPLE 6

3'-O-(2-Nitrobenzyl)-6'-O-(3,4-diaza-5-oxoeicos-2-en-1-yl)fluorescein(18).

The aldehyde (8) was prepared by ozonolysis of 2-nitrobenzyl allylfluorescein (3; 51 mg) as described in Example 3, and mixed with asolution of palmitic hydrazide (40 mg, prepared as described by Curtiusand Dellschaft⁸) in ethanol (0.9 ml) and tetrahydrofuran (0.3 ml). Thesolution was heated under reflux for 1 h and the solvent evaporatedunder reduced pressure. The residue was purified by flash chromatography(ethyl acetate--CH₂ Cl₂ 15:85) to afford the lipophilic hydrazone (18)as a colourless glass (46 mg) which was characterised by ¹ H-NMRspectroscopy.

The product has the following formula: ##STR10## This is a cagedfluorescein compound with a highly lipophilic nature and is suitable foruse in the labelling of lipid bilayers or other lipid structures.

1. C. D. Hurd and L. Schmerling, J. Am. Chem. Soc., 1937, 59, 113.

2. G. A. Krafft, W. R. Sutton and R. T. Cummings, J. Am. Chem. Soc.,1988, 110, 301.

3. W. Streicher and H. Reinshagen, Chem. Ber., 1975, 108, 813.

4. D. H. Rich, P. D. Gesellchen, A. Tong, A. Chueng and C. K. Buchner,J. Med. Chem., 1975, 18, 1004.

5. L. Paul, A. Dittmar and C. Rusch, Chem. Ber., 1967, 100, 2757.

6. C. D. Ritchie, J. A. Wenniger and J. H. Jones, J. Ass. Off. Agric.Chem., 1959, 42, 720.

7. H. Sensui, M. Gonda and T. Obara, Eur. Patent Appl. 184 114.

8. T. Curtius and F. H. Dellschaft, J. Prakt. Chem., 1901, 64, 419.

BRIEF DESCRIPTION OF DRAWING

Lanes 2 and 7 contain equal masses of protein, but the LC-1 in lane 2(incubate A in Example 4) had been pretreated with N-ethylmaleimideprior to treatment with the caged fluorescein-maleimide. The LC-1 inlane 7 is derived from incubate B and shows specific incorporation ofthe caged fluorescein. As described in the labelled protein preparationswere irradiated prior to electrophoresis to release the fluorescence ofthe caged species, and the gel was photographed under ultravioletirradiation to visualise fluorescent species.

We claim:
 1. A compound having the following formula: ##STR11## whereinX is an optionally substituted benzyl group which carries a NO₂ group inthe ortho-position,Z is a group of the formula: ##STR12## m and n areeach integers of from 1 to 6, and q is an integer of from 1 to 20,andsalts thereof.
 2. A compound as claimed in claim 1 of the formula:##STR13##
 3. A method for preparing a compound (I) as claimed in claim1, which comprises the following sequence of reactions: ##STR14##wherein X is an optionally substituted benzyl group which carries a NO₂group in the ortho-position, andm and n are each integers of from 1 to6, and salts thereof.